(adapted from abstract) The heat shock protein Hsp72 is a molecular chaperone that refolds damaged proteins and protects the cell from various stresses. In addition, Hsp72 was shown to play an unexpected role as a specific regulator of the cell death program. Findings from this Laboratory show that a critical event in the Hsp72-mediated prevention of apoptosis is suppression of a protein kinase JNK. Hsp72 inhibits JNK through stimulation of an unidentified JNK phosphatase. A cell's decision to either undergo apoptosis upon exposure to stresses, or to repair thus appears to depend on a fine balance between the induction of Hsp72 and the activation of JNK. Understanding the detailed mechanism of this cell fate control is the focus of the present proposal. The first goal of the proposed research is to establish the mechanism of Hsp72-mediated regulation of JNK dephosphorylation. Findings from this Laboratory led to a model that sequestering of Hsp72 by abnormal proteins generated in the cytosol as a result of heat shock and other protein damaging stresses is a primary event in JNK regulation. It will be established whether association with Hsp72 is critical for the ability of abnormal polypeptides to activate JNK; whether JNK activation induced by abnormal polypeptides proceeds via inhibition of JNK dephosphorylation; and whether other heat shock proteins which cooperate with Hsp72 in protein refolding are involved in JNK regulation. The second goal is to identify and characterize JNK phosphatase, which is regulated by heat shock and by Hsp72. The possible function of a phosphatase Shp-2 as a JNK phosphatase will be studied. An additional major JNK dephosphorylating activity will be purified. The enzyme will be identified and cloned. The role of this enzyme in dephosphorylating JNK in viva will be probed by a variety of approaches, such as ectopic overexpression of the normal gene and a dominant negative mutant; and expressing an antisense RNA to inhibit phosphatase translation. Regulation of this enzyme by heat shock and Hsp72 will be investigated. Elucidating the mechanisms of JNK suppression by Hsp72 is essential for understanding of heat resistance of cells, an important factor in hyperthermia treatment of cancer patients. Furthermore, similar mechanisms may function in Hsp72-mediated protection of cells from genotoxic stresses and anti-cancer drugs. Results of this research may suggest whether JNK phosphatase and Hsp72 could be attractive new targets for anticancer drug design.